The long term goal of this research program is to define the role of the Origin Recognition Complex (ORC) in cell cycle progression in eukaryotes. The ORC is a critical component for DMA replication, but also has a role in other cell functions. In particular, we have recently demonstrated that the complex is critically involved in cytokinesis. We found that the smallest ORC subunit, Orc6, is localized to the cell membranes and cleavage furrows of dividing cells. Orc6 interacts with the septin protein Peanut (Pnut) and is involved in cytokinesis in addition to its replication functions. The following specific aims will be pursued: 1) molecular analysis of Orc6-Pnut interaction. The function of conserved sequence motifs of Orc6 and Pnut proteins will be defined and the role of Pnut in Orc6 localization will be determined. 2) Determining the motifs and sequences of Orc6 that are important for DNA binding and replication. Critical residues of Orc6 essential for DNA binding and replication will be identified and their importance confirmed both in vitro and in vivo. 3) Analysis of the intracellular localization of Orc6 during the cell cycle. Functional significance of Orc6 localization during different stages of the cell cycle will be studied. 4) Targeted mutagenesis of Orc6 in vivo and analysis of the loss of function phenotype. Targeted mutagenesis will be used as well as other methods that allow us to mimic the mutant phenotype to study Orc6 functions in vivo. Working with Drosophila will be advantageous for these studies as it allows the use of both biochemical and genetic methods and also permits insights into the developmental as well as tissue specific aspects of replication control. An understanding of the molecular events involved in the initiation of replication and cytokinesis will provide a basis for ultimately controlling these processes. Such control might lead to rationally designed therapies for cancer and may provide the means for regenerating cells lost due to aging, disease or injury.